2-amino-n- [7-methoxy-2, 3-dihydroimidazo-[1, 2-c] quinazolin-5-yl] pyrimidine-5-carboxamides

ABSTRACT

This invention relates to novel 2,3-dihydroimidazo[1,2-c]quinazoline compounds, pharmaceutical compositions containing such compounds and the use of those compounds or compositions for phosphotidylinositol-3-kinase (PI3K) inhibition and treating diseases associated with phosphotidylinositol-3-kinase (PI3K) activity, in particular treating hyper-proliferative and/or angiogenesis disorders, as a sole agent or in combination with other active ingredients.

FIELD OF THE INVENTION

This invention relates to novel 2,3-dihydroimidazo[1,2-c]quinazolinecompounds, pharmaceutical compositions containing such compounds and theuse of those compounds or compositions for phosphotidylinositol-3-kinase(PI3K) inhibition and treating diseases associated withphosphotidylinositol-3-kinase (PI3K) activity, in particular treatinghyper-proliferative and/or angiogenesis disorders, as a sole agent or incombination with other active ingredients.

BACKGROUND OF THE INVENTION

In the last decade the concept of developing anti-cancer medicationswhich target abnormally active protein kinases has led to a number ofsuccesses. In addition to the actions of protein kinases, lipid kinasesalso play an important role in generating critical regulatory secondmessengers. The PI3K family of lipid kinases generates3′-phosphoinositides that bind to and activate a variety of cellulartargets, initiating a wide range of signal transduction cascades(Vanhaesebroeck et al., 2001; Toker, 2002; Pendaries et al., 2003;Downes et al., 2005). These cascades ultimately induce changes inmultiple cellular processes, including cell proliferation, cellsurvival, differentiation, vesicle trafficking, migration, andchemotaxis.

PI3Ks can be divided into three distinct classes based upon differencesin both structure, and substrate preference. While members of the ClassII family of PI3Ks have been implicated in the regulation of tumorgrowth (Brown and Shepard, 2001; Traer et al., 2006), the bulk ofresearch has focused on the Class I enzymes and their role in cancer(Vivanco And Sawyers, 2002; Workman, 2004, Chen et al., 2005; Hennesseyet al., 2005; Stauffer et al., 2005; Stephens et al., 2005; Cully etal., 2006).

Class I PI3Ks have traditionally been divided into two distinctsub-classes based upon differences in protein subunit composition. TheClass I_(A) PI3Ks are comprised of a catalytic p110 catalytic subunit(p110α, β or δ) heterodimerized with a member of the p85 regulatorysubunit family. In contrast, the Class I_(B) PI3K catalytic subunit(p110γ) heterodimerizes with a distinct p101 regulatory subunit(reviewed by Vanhaesebroeck and Waterfield, 1999; Funaki et al., 2000;Katso et al., 2001). The C-terminal region of these proteins contains acatalytic domain that possesses distant homology to protein kinases. ThePI3Kγ structure is similar to Class I_(A) p110s, but lacks theN-terminal p85 binding site (Domin and Waterfield, 1997). Though similarin overall structure, the homology between catalytic p110 subunits islow to moderate. The highest homology between the PI3K isoforms is inthe kinase pocket of the kinase domain.

The Class I_(A) PI3K isoforms associate with activated receptor tyrosinekinases (RTKs) (including PDGFR, EGFR, VEGFR, IGF1-R, c-KIT, CSF-R andMet), or with tyrosine phosphorylated adapter proteins (such as Grb2,Cbl, IRS-1 or Gab1), via their p85 regulatory subunits resulting instimulation of the lipid kinase activity. Activation of the lipid kinaseactivity of the p110β and p110γ isoforms has been shown to occur inresponse to binding to activated forms of the ras Oncogene (Kodaki etal, 1994). In fact, the oncogenic activity of these isoforms may requirebinding to ras (Kang et al., 2006). In contrast, the p110α and p110δisoforms exhibit oncogenic activity independent of ras binding, throughconstitutive activation of Akt.

Class I PI3Ks catalyze the conversion of PI(4,5)P₂ [PIP₂] to PI(3,4,5)P₃[PIP₃]. The production of PIP₃ by PI3K affects multiple signalingprocesses that regulate and coordinate the biological end points of cellproliferation, cell survival, differentiation and cell migration. PIP₃is bound by Pleckstrin-Homology (PH) domain-containing proteins,including the phosphoinositide-dependent kinase, PDK1 and the Aktproto-oncogene product, localizing these proteins in regions of activesignal transduction and also contributing directly to their activation(Klippel et al., 1997; Fleming et al., 2000; Itoh and Takenawa, 2002;Lemmon, 2003). This co-localization of PDK1 with Akt facilitates thephosphorylation and activation of Akt. Carboxy-terminal phosphorylationof Akt on Ser⁴⁷³ promotes phosphorylation of Thr³⁰⁸ in the Aktactivation loop (Chan and Tsichlis, 2001; Hodgekinson et al., 2002;Scheid et al., 2002; Hresko et al., 2003). Once active, Aktphosphorylates and regulates multiple regulatory kinases of pathwaysthat directly influence cell cycle progression and cell survival.

Many of the effects of Akt activation are mediated via its negativeregulation of pathways which impact cell survival and which are commonlydysregulated in cancer. Akt promotes tumor cell survival by regulatingcomponents of the apoptotic and cell cycle machinery. Akt is one ofseveral kinases that phosphorylate and inactivate pro-apoptotic BADproteins (del Paso et al., 1997; Pastorino et al., 1999). Akt may alsopromote cell survival through blocking cytochrome C-dependent caspaseactivation by phosphorylating Caspase 9 on Ser¹⁹⁶ (Cardone et al.,1998).

Akt impacts gene transcription on several levels. The Akt-mediatedphosphorylation of the MDM2 E3 ubiquitin ligase on Ser¹⁶⁶ and Ser¹⁸⁶facilitates the nuclear import of MDM2 and the formation and activationof the ubiquitin ligase complex. Nuclear MDM2 targets the p53 tumorsuppressor for degradation, a process that can be blocked by LY294002(Yap et al., 2000; Ogarawa et al., 2002). Downregulation of p53 by MDM2negatively impacts the transcription of p53-regulated pro-apoptoticgenes (e.g. Bax, Fas, PUMA and DR5), the cell cycle inhibitor,p21^(Cip1), and the PTEN tumor suppressor (Momand et al., 2000; Hupp etal., 2000; Mayo et al., 2002; Su et al., 2003). Similarly, theAkt-mediated phosphorylation of the Forkhead transcription factors FKHR,FKHRL and AFX (Kops et al., 1999; Tang et al., 1999), facilitates theirbinding to 14-3-3 proteins and export from the cell nucleus to thecytosol (Brunet et al., 1999). This functional inactivation of Forkheadactivity also impacts pro-apoptotic and pro-angiogenic genetranscription including the transcription of Fas ligand (Ciechomska etal., 2003) Bim, a pro-apoptotic Bcl-2 family member (Dijkers et al.,2000), and the Angiopoietin-1 (Ang-1) antagonist, Ang-2 (Daly et al.,2004). Forkhead transcription factors regulate the expression of thecyclin-dependent kinase (Cdk) inhibitor p27^(Kip1). Indeed, PI3Kinhibitors have been demonstrated to induce p27^(Kip1) expressionresulting in Cdk1 inhibition, cell cycle arrest and apoptosis (Dijkerset al., 2000). Akt is also reported to phosphorylate p21^(Cip1) onThr¹⁴⁵ and p27^(Kip1) on Thr¹⁵⁷ facilitating their association with14-3-3 proteins, resulting in nuclear export and cytoplasmic retention,preventing their inhibition of nuclear Cdks (Zhou et al., 2001; Motti etal., 2004; Sekimoto et al., 2004). In addition to these effects, Aktphosphorylates IKK (Romashkova and Makarov, 1999), leading to thephosphorylation and degradation of IκB and subsequent nucleartranslocation of NFκB, resulting in the expression of survival genessuch as IAP and Bcl-X_(L).

The PI3K/Akt pathway is also linked to the suppression of apoptosisthrough the JNK and p38^(MAPK) MAP Kinases that are associated with theinduction of apoptosis. Akt is postulated to suppress JNK and p38^(MAPK)signaling through the phosphorylation and inhibition of two JNK/p38regulatory kinases, Apoptosis Signal-regulating Kinase 1 (ASK1) (Kim etal., 2001: Liao and Hung, 2003; Yuan et al., 2003), and Mixed LineageKinase 3 (MLK3) (Lopez-llasaca et al., 1997; Barthwal et al., 2003;Figueroa et al., 2003;). The induction of p38^(MAPK) activity isobserved in tumors treated with cytotoxic agents and is required forthose agents to induce cell death (reviewed by Olson and Hallahan,2004). Thus, inhibitors of the PI3K pathway may promote the activitiesof co-administered cytotoxic drugs.

An additional role for PI3K/Akt signaling involves the regulation ofcell cycle progression through modulation of Glycogen Synthase Kinase 3(GSK3) activity. GSK3 activity is elevated in quiescent cells, where itphosphorylates cyclin D₁ on Ser²⁸⁶, targeting the protein forubiquitination and degradation (Diehl et al., 1998) and blocking entryinto S-phase. Akt inhibits GSK3 activity through phosphorylation on Ser⁹(Cross et al., 1995). This results in the elevation of Cyclin D₁ levelswhich promotes cell cycle progression. Inhibition of GSK3 activity alsoimpacts cell proliferation through activation of the wnt/beta-cateninsignaling pathway (Abbosh and Nephew, 2005; Naito et al., 2005; Wilkeret al., 2005; Kim et al., 2006; Segrelles et al., 2006). Akt mediatedphosphorylation of GSK3 results in stabilization and nuclearlocalization of the beta-catenin protein, which in turn leads toincreased expression of c-myc and cyclin D1, targets of thebeta-catenin/Tcf pathway.

Although PI3K signaling is utilized by many of the signal transductionnetworks associated with both oncogenes and tumor suppressors, PI3K andits activity have been linked directly to cancer. Overexpression of boththe p110α and p110β isoforms has been observed in bladder and colontumors and cell lines, and overexpression generally correlates withincreased PI3K activity (Benistant et al., 2000). Overexpression ofp110α has also been reported in ovarian and cervical tumors and tumorcell lines, as well as in squamous cell lung carcinomas. Theoverexpression of p110α in cervical and ovarian tumor lines isassociated with increased PI3K activity (Shayesteh et al., 1999; Ma etal., 2000). Elevated PI3K activity has been observed in colorectalcarcinomas (Phillips et al., 1998) and increased expression has beenobserved in breast carcinomas (Gershtein et al., 1999).

Over the last few years, somatic mutations in the gene encoding p110α(PIK3CA) have been identified in numerous cancers. The data collected todate suggests that PIK3CA is mutated in approximately 32% of colorectalcancers (Samuels et al., 2004; Ikenoue et al., 2005), 18-40% of breastcancers (Bachman et al., 2004; Campbell et al., 2004; Levine et al.,2005; Saal et al., 2005; Wu et al., 2005), 27% of glioblastomas (Samuelset al., 2004; Hartmann et al., 2005, Gallia et al., 2006), 25% ofgastric cancers (Byun et al., 2003; Samuels et al., 2004; Li et al.,2005), 36% of hepatocellular carcinomas (Lee et al., 2005), 4-12% ofovarian cancers (Levine et al., 2005; Wang et al., 2005), 4% of lungcancers (Samuels et al., 2004; Whyte and Holbeck, 2006), and up to 40%of endometrial cancers (Oda et al., 2005). PIK3CA mutations have beenreported in oligodendroma, astrocytoma, medulloblastoma, and thyroidtumors as well (Broderick et al., 2004; Garcia-Rostan et al., 2005).Based upon the observed high frequency of mutation, PIK3CA is one of thetwo most frequently mutated genes associated with cancer, the otherbeing K-ras. More than 80% of the PIK3CA mutations cluster within tworegions of the protein, the helical (E545K) and catalytic (H1047R)domains. Biochemical analysis and protein expression studies havedemonstrated that both mutations lead to increased constitutive p110αcatalytic activity and are in fact, oncogenic (Bader et al., 2006; Kanget al., 2005; Samuels et al., 2005; Samuels and Ericson, 2006).Recently, it has been reported that PIK3CA knockout mouse embryofibroblasts are deficient in signaling downstream from various growthfactor receptors (IGF-1, Insulin, PDGF, EGF), and are resistant totransformation by a variety of oncogenic RTKs (IGFR, wild-type EGFR andsomatic activating mutants of EGFR, Her2/Neu)(Zhao et al., 2006).

Functional studies of PI3K in vivo have demonstrated that siRNA-mediateddownregulation of p110β inhibits both Akt phosphorylation and HeLa celltumor growth in nude mice (Czauderna et al., 2003). In similarexperiments, siRNA-mediated downregulation of p110β was also shown toinhibit the growth of malignant glioma cells in vitro and in vivo (Pu etal., 2006). Inhibition of PI3K function by dominant-negative p85regulatory subunits can block mitogenesis and cell transformation (Huanget al., 1996; Rahimi et al., 1996). Several somatic mutations in thegenes encoding the p85a and p85β regulatory subunits of PI3K that resultin elevated lipid kinase activity have been identified in a number ofcancer cells as well (Janssen et al., 1998; Jimenez et al., 1998; Philpet al., 2001; Jucker et al., 2002; Shekar et al., 2005). NeutralizingPI3K antibodies also block mitogenesis and can induce apoptosis in vitro(Roche et al., 1994: Roche et al., 1998; Benistant et al., 2000). Invivo proof-of-principle studies using the PI3K inhibitors LY294002 andwortmannin, demonstrate that inhibition of PI3K signaling slows tumorgrowth in vivo (Powis et al., 1994; Shultz et al., 1995; Semba et al.,2002; Ihle et al., 2004).

Overexpression of Class I PI3K activity, or stimulation of their lipidkinase activities, is associated with resistance to both targeted (suchas imatinib and tratsuzumab) and cytotoxic chemotherapeutic approaches,as well as radiation therapy (West et al., 2002; Gupta et al., 2003;Osaki et al., 2004; Nagata et al., 2004; Gottschalk et al., 2005; Kim etal., 2005). Activation of PI3K has also been shown to lead to expressionof multidrug resistant protein-1 (MRP-1) in prostate cancer cells andthe subsequent induction of resistance to chemotherapy (Lee et al.,2004).

The importance of PI3K signaling in tumorigenesis is further underscoredby the findings that the PTEN tumor suppressor, a PI(3)P phosphatase, isamong the most commonly inactivated genes in human cancers (Li et al.,1997, Steck et al., 1997; Ali et al., 1999; Ishii et al., 1999). PTENdephosphorylates PI(3,4,5)P₃ to PI(4,5)P₂ thereby antagonizingPI3K-dependent signaling. Cells containing functionally inactive PTENhave elevated levels of PIP₃, high levels of activity of PI3K signaling(Haas-Kogan et al., 1998; Myers et al., 1998; Taylor et al., 2000),increased proliferative potential, and decreased sensitivity topro-apoptotic stimuli (Stambolic et al., 1998). Reconstitution of afunctional PTEN suppresses PI3K signaling (Taylor et al., 2000),inhibits cell growth and re-sensitizes cells to pro-apoptotic stimuli(Myers et al., 1998; Zhao et al., 2004). Similarly, restoration of PTENfunction in tumors lacking functional PTEN inhibits tumor growth in vivo(Stahl et al., 2003; Su et al., 2003; Tanaka and Grossman, 2003) andsensitizes cells to cytotoxic agents (Tanaka and Grossman, 2003).

The class I family of PI3Ks clearly plays an important role in theregulation of multiple signal transduction pathways that promote cellsurvival and cell proliferation, and activation of their lipid kinaseactivity contributes significantly to the development of humanmalignancies. Furthermore, inhibition of PI3K may potentially circumventthe cellular mechanisms that underlie resistance to chemotherapeuticagents. A potent inhibitor of Class I PI3K activities would thereforehave the potential not only to inhibit tumor growth but to alsosensitize tumor cells to pro-apoptotic stimuli in vivo.

Signal transduction pathways originating from chemoattractant receptorsare considered to be important targets in controlling leukocyte motilityin inflammatory diseases. Leukocyte trafficking is controlled bychemoattractant factors that activate heterotrimeric GPCRs and therebytrigger a variety of downstream intracellular events. Signaltransduction along one of these pathways that results in mobilization offree Ca²⁺, cytoskeletal reorganization, and directional movement dependson lipid-derived second messengers produced by PI3K activity (Wymann etal., 2000; Stein and Waterfield, 2000).

PI3Kγ modulates baseline cAMP levels and controls contractility incells. Recent research indicates that alterations in baseline cAMPlevels contributes to the increased contractility in mutant mice. Thisresearch, therefore, shows that PI3Kγ inhibitors would afford potentialtreatments for congestive heart failure, ischemia, pulmonaryhypertension, renal failure, cardiac hypertrophy, atherosclerosis,thromboembolism, and diabetes.

PI3K inhibitors would be expected to block signal transduction fromGPCRs and block the activation of various immune cells, leading to abroad anti-inflammatory profile with potential for the treatment ofinflammatory and immunoregulatory diseases, including asthma, atopicdermatitis, rhinitis, allergic diseases, chronic obstructive pulmonarydisease (COPD), septic shock, joint diseases, autoimmune pathologiessuch as rheumatoid arthritis and Graves' disease, diabetes, cancer,myocardial contractility disorders, thromboembolism, andatherosclerosis.

2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamide(10), (which is hereinafter referred to as “copanlisib”), is aproprietary cancer agent with a novel mechanism of action, inhibitingClass I phosphatidylinositol-3-kinases (PI3Ks). This class of kinases isan attractive target since PI3Ks play a central role in the transductionof cellular signals from surface receptors for survival andproliferation. Copanlisib exhibits a broad spectrum of activity againsttumours of multiple histologic types, both in vitro and in vivo.

Copanlisib may be synthesised according to the methods given ininternational patent application PCT/EP2003/010377, published as WO04/029055 A1 on Apr. 8, 2004, (which is incorporated herein by referencein its entirety), on pp. 26 et seq.

Copanlisib is published in international patent applicationPCT/US2007/024985, published as WO 2008/070150 A1 on Jun. 12, 2008,(which is incorporated herein by reference in its entirety), as thecompound of Example13:2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamide.

Copanlisib may be synthesized according to the methods given in WO2008/070150, pp. 9 et seq., and on pp. 42 et seq. Biological test datafor said compound of formula (I) is given in WO 2008/070150 on pp. 101to 107.

2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimid-azo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamidedihydrochloride (11), (which is hereinafter referred to as “copanlisibdihydrochloride”) is published in international patent applicationPCT/EP2012/055600, published as WO 2012/136553 on Oct. 11, 2012, (whichis incorporated herein by reference in its entirety), as the compound ofExamples 1 and2:2-amino-N-[7-methoxy-8-(3-morpholin-4-ylpropoxy)-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamidedihydrochloride: it may be synthesized according to the methods given insaid Examples 1 and 2.

However, the state of the art described above does not describe thespecific2-amino-N-[7-methoxy-2,3-dihydroimidazo-[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamidesas described herein, or a stereoisomer, a tautomer, an N-oxide, ahydrate, a solvate, or a salt thereof, or a mixture of same, asdescribed and defined herein, and as hereinafter referred to as“compounds of the present invention”, or their pharmacological activity.

It has now been found, and this constitutes the basis of the presentinvention, that said compounds of the present invention have surprisingand advantageous properties, in particular they exhibitanti-proliferative activity and are thus useful to prevent or treat thedisorders associated with hyper-proliferation.

DESCRIPTION OF THE INVENTION

According to a first aspect, the present invention relates to compoundsof general formula (I):

in which:R¹ represents a hydrogen atom or a —C(═O)H group; andR^(1′) represents a —(CH₂)₂OH group;or:R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with 1 or 2 substituentsindependently selected from —OH and ═O;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ represents a hydrogen atom; andR^(1′) represents a —(CH₂)₂OH group;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ represents a —C(═O)H group; andR^(1′) represents a —(CH₂)₂OH group;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with 1 or 2 substituentsindependently selected from —OH and ═O;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with a —OH and a ═Osubstituent;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with a —OH substituent;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with a ═O substituent;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In an embodiment of the first aspect, the present invention relates tocompounds of general formula (I), in which:

R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with a ═O substituent, said ═Ogroup being bound to a carbon atom adjacent to said N-atom;or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In another embodiment of the first aspect, the present invention relatesto compounds of formula (I), which are selected from:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In another embodiment of the first aspect, the present invention relatesto a compound of formula (I), which is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In another embodiment of the first aspect, the present invention relatesto a compound of formula (I), which is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In another embodiment of the first aspect, the present invention relatesto a compound of formula (I), which is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In another embodiment of the first aspect, the present invention relatesto a compound of formula (I), which is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

In another embodiment of the first aspect, the present invention relatesto a compound of formula (I), which is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof.

Where there is a discrepancy between the chemical name and the chemicalstructure depicted, the chemical structure depicted takes precedenceover the chemical name given.

Without being bound by theory or mechanism, the compounds of the presentinvention display surprising activity for the inhibition ofphosphatidylinositol-3-kinase and chemical and structural stability overthose compounds of the prior art, especially since it was believed thatactivity is based on the chemical structure of the compounds, inparticular the basicity of the compounds as a result of R¹ being aminooptionally substituted with R⁵ and R^(5′).

Definitions

Where the plural form of the word compounds, salts, polymorphs,hydrates, solvates and the like, is used herein, this is taken to meanalso a single compound, salt, polymorph, isomer, hydrate, solvate or thelike.

The compounds of this invention may contain one or more asymmetriccenters, depending upon the location and nature of the varioussubstituents desired. Asymmetric carbon atoms may be present in the (R)or (S) configuration, resulting in racemic mixtures in the case of asingle asymmetric center, and diastereomeric mixtures in the case ofmultiple asymmetric centers. In certain instances, asymmetry may also bepresent due to restricted rotation about a given bond, for example, thecentral bond adjoining two substituted aromatic rings of the specifiedcompounds. Substituents on a ring may also be present in either cis ortrans form. It is intended that all such configurations (includingenantiomers and diastereomers), are included within the scope of thepresent invention. Preferred compounds are those, which produce the moredesirable biological activity. Separated, pure or partially purifiedisomers and stereoisomers or racemic or diastereomeric mixtures of thecompounds of this invention are also included within the scope of thepresent invention. The purification and the separation of such materialscan be accomplished by standard techniques known in the art.

The present invention also relates to useful forms of the compounds asdisclosed herein, such as pharmaceutically acceptable salts,co-precipitates, metabolites, hydrates, solvates and prodrugs of all thecompounds of examples. Further, it is possible for the compounds of thepresent invention to exist in free form, e.g. as a free base, or as afree acid, or as a zwitterion, or to exist in the form of a salt. Saidsalt may be any salt, either an organic or inorganic addition salt,particularly any pharmaceutically acceptable organic or inorganicaddition salt, which is customarily used in pharmacy, or which is used,for example, for isolating or purifying the compounds of the presentinvention.

The term “pharmaceutically acceptable salt” refers to an inorganic ororganic acid addition salt of a compound of the present invention. Forexample, see S. M. Berge, et al. “Pharmaceutical Salts,” J. Pharm. Sci.1977, 66, 1-19.

A suitable pharmaceutically acceptable salt of the compounds of thepresent invention may be, for example, an acid-addition salt of acompound of the present invention bearing a nitrogen atom, in a chain orin a ring, for example, which is sufficiently basic, such as anacid-addition salt with an inorganic acid, or “mineral acid”, such ashydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, bisulfuric,phosphoric, or nitric acid, for example, or with an organic acid, suchas formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic,butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic,2-(4-hydroxybenzoyl)-benzoic, camphoric, cinnamic,cyclopentanepropionic, digluconic, 3-hydroxy-2-naphthoic, nicotinic,pamoic, pectinic, 3-phenylpropionic, pivalic, 2-hydroxyethanesulfonic,itaconic, trifluoromethanesulfonic, dodecylsulfuric, ethanesulfonic,benzenesulfonic, para-toluenesulfonic, methanesulfonic,2-naphthalenesulfonic, naphthalinedisulfonic, camphorsulfonic acid,citric, tartaric, stearic, lactic, oxalic, malonic, succinic, malic,adipic, alginic, maleic, fumaric, D-gluconic, mandelic, ascorbic,glucoheptanoic, glycerophosphoric, aspartic, sulfosalicylic, orthiocyanic acid, for example.

Further, another suitably pharmaceutically acceptable salt of a compoundof the present invention which is sufficiently acidic, is an alkalimetal salt, for example a sodium or potassium salt, an alkaline earthmetal salt, for example a calcium, magnesium or strontium salt, or analuminium or a zinc salt, or an ammonium salt derived from ammonia orfrom an organic primary, secondary or tertiary amine having 1 to 20carbon atoms, such as ethylamine, diethylamine, triethylamine,ethyldiisopropylamine, monoethanolamine, diethanolamine,triethanolamine, dicyclohexylamine, dimethylaminoethanol,diethylaminoethanol, tris(hydroxymethyl)aminomethane, procaine,dibenzylamine, N-methylmorpholine, arginine, lysine,1,2-ethylenediamine, N-methylpiperidine, N-methyl-glucamine,N,N-dimethyl-glucamine, N-ethyl-glucamine, 1,6-hexanediamine,glucosamine, sarcosine, serinol, 2-amino-1,3-propanediol,3-amino-1,2-propanediol, 4-amino-1,2,3-butanetriol, or a salt with aquarternary ammonium ion having 1 to 20 carbon atoms, such astetramethylammonium, tetraethylammonium, tetra(n-propyl)ammonium,tetra(n-butyl)ammonium, N-benzyl-N,N,N-trimethylammonium, choline orbenzalkonium.

Those skilled in the art will further recognise that it is possible foracid addition salts of the claimed compounds to be prepared by reactionof the compounds with the appropriate inorganic or organic acid via anyof a number of known methods. Alternatively, alkali and alkaline earthmetal salts of acidic compounds of the present invention are prepared byreacting the compounds of the present invention with the appropriatebase via a variety of known methods.

The present invention includes all possible salts of the compounds ofthe present invention as single salts, or as any mixture of said salts,in any ratio.

In the present text, in particular in the Experimental Section, for thesynthesis of intermediates and of examples of the present invention,when a compound is mentioned as a salt form with the corresponding baseor acid, the exact stoichiometric composition of said salt form, asobtained by the respective preparation and/or purification process, is,in most cases, unknown.

Unless specified otherwise, suffixes to chemical names or structuralformulae relating to salts, such as “hydrochloride”, “trifluoroacetate”,“sodium salt”, or “x HCl”, “x CF₃COOH”, “x Na⁺”, for example, mean asalt form, the stoichiometry of which salt form not being specified.

This applies analogously to cases in which synthesis intermediates orexample compounds or salts thereof have been obtained, by thepreparation and/or purification processes described, as solvates, suchas hydrates, with (if defined) unknown stoichiometric composition.

A solvate for the purpose of this invention is a complex of a solventand a compound of the invention in the solid state. Exemplary solvateswould include, but are not limited to, complexes of a compound of theinvention with ethanol or methanol. Hydrates are a specific form ofsolvate wherein the solvent is water.

Pharmaceutical Compositions of the Compounds of the Invention

This invention also relates to pharmaceutical compositions containingone or more compounds of the present invention.

It is possible for the compounds according to the invention to havesystemic and/or local activity. For this purpose, they can beadministered in a suitable manner, such as, for example, via the oral,parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal,vaginal, dermal, transdermal, conjunctival, otic route or as an implantor stent.

For these administration routes, it is possible for the compoundsaccording to the invention to be administered in suitable administrationforms.

For oral administration, it is possible to formulate the compoundsaccording to the invention to dosage forms known in the art that deliverthe compounds of the invention rapidly and/or in a modified manner, suchas, for example, tablets (uncoated or coated tablets, for example withenteric or controlled release coatings that dissolve with a delay or areinsoluble), orally-disintegrating tablets, films/wafers,films/lyophylisates, capsules (for example hard or soft gelatinecapsules), sugar-coated tablets, granules, pellets, powders, emulsions,suspensions, aerosols or solutions. It is possible to incorporate thecompounds according to the invention in crystalline and/or amorphisedand/or dissolved form into said dosage forms.

Parenteral administration can be effected with avoidance of anabsorption step (for example intravenous, intraarterial, intracardial,intraspinal or intralumbal) or with inclusion of absorption (for exampleintramuscular, subcutaneous, intracutaneous, percutaneous orintraperitoneal). Administration forms which are suitable for parenteraladministration are, inter alia, preparations for injection and infusionin the form of solutions, suspensions, emulsions, lyophylisates orsterile powders.

Examples which are suitable for other administration routes arepharmaceutical forms for inhalation [inter alia powder inhalers,nebulizers], nasal drops, nasal solutions, nasal sprays;tablets/films/wafers/capsules for lingual, sublingual or buccaladministration; suppositories; eye drops, eye ointments, eye baths,ocular inserts, ear drops, ear sprays, ear powders, ear-rinses, eartampons; vaginal capsules, aqueous suspensions (lotions, mixturaeagitandae), lipophilic suspensions, emulsions, ointments, creams,transdermal therapeutic systems (such as, for example, patches), milk,pastes, foams, dusting powders, implants or stents.

The compounds according to the invention can be incorporated into thestated administration forms. This can be effected in a manner known perse by mixing with pharmaceutically suitable excipients. Pharmaceuticallysuitable excipients include, inter alia,

-   -   fillers and carriers (for example cellulose, microcrystalline        cellulose (such as, for example, Avicel®), lactose, mannitol,        starch, calcium phosphate (such as, for example, Di-Cafos®)),    -   ointment bases (for example petroleum jelly, paraffins,        triglycerides, waxes, wool wax, wool wax alcohols, lanolin,        hydrophilic ointment, polyethylene glycols),    -   bases for suppositories (for example polyethylene glycols, cacao        butter, hard fat),    -   solvents (for example water, ethanol, isopropanol, glycerol,        propylene glycol, medium chain-length triglycerides fatty oils,        liquid polyethylene glycols, paraffins),    -   surfactants, emulsifiers, dispersants or wetters (for example        sodium dodecyl sulfate), lecithin, phospholipids, fatty alcohols        (such as, for example, Lanette®), sorbitan fatty acid esters        (such as, for example, Span®), polyoxyethylene sorbitan fatty        acid esters (such as, for example, Tween®), polyoxyethylene        fatty acid glycerides (such as, for example, Cremophor®),        polyoxethylene fatty acid esters, polyoxyethylene fatty alcohol        ethers, glycerol fatty acid esters, poloxamers (such as, for        example, Pluronic®),    -   buffers, acids and bases (for example phosphates, carbonates,        citric acid, acetic acid, hydrochloric acid, sodium hydroxide        solution, ammonium carbonate, trometamol, triethanolamine),    -   isotonicity agents (for example glucose, sodium chloride),    -   adsorbents (for example highly-disperse silicas),    -   viscosity-increasing agents, gel formers, thickeners and/or        binders (for example polyvinylpyrrolidone, methylcellulose,        hydroxypropylmethylcellulose, hydroxypropylcellulose,        carboxymethylcellulose-sodium, starch, carbomers, polyacrylic        acids (such as, for example, Carbopol®); alginates, gelatine),    -   disintegrants (for example modified starch,        carboxymethylcellulose-sodium, sodium starch glycolate (such as,        for example, Explotab®), cross-linked polyvinylpyrrolidone,        croscarmellose-sodium (such as, for example, AcDiSol®)),    -   flow regulators, lubricants, glidants and mould release agents        (for example magnesium stearate, stearic acid, talc,        highly-disperse silicas (such as, for example, Aerosil®)),    -   coating materials (for example sugar, shellac) and film formers        for films or diffusion membranes which dissolve rapidly or in a        modified manner (for example polyvinylpyrrolidones (such as, for        example, Kollidon®), polyvinyl alcohol,        hydroxypropylmethylcellulose, hydroxypropylcellulose,        ethylcellulose, hydroxypropylmethylcellulose phthalate,        cellulose acetate, cellulose acetate phthalate, polyacrylates,        polymethacrylates such as, for example, Eudragit®)),    -   capsule materials (for example gelatine,        hydroxypropylmethylcellulose),    -   synthetic polymers (for example polylactides, polyglycolides,        polyacrylates, polymethacrylates (such as, for example,        Eudragit®), polyvinylpyrrolidones (such as, for example,        Kollidon®), polyvinyl alcohols, polyvinyl acetates, polyethylene        oxides, polyethylene glycols and their copolymers and        blockcopolymers),    -   plasticizers (for example polyethylene glycols, propylene        glycol, glycerol, triacetine, triacetyl citrate, dibutyl        phthalate),    -   penetration enhancers,    -   stabilisers (for example antioxidants such as, for example,        ascorbic acid, ascorbyl palmitate, sodium ascorbate,        butylhydroxyanisole, butylhydroxytoluene, propyl gallate),    -   preservatives (for example parabens, sorbic acid, thiomersal,        benzalkonium chloride, chlorhexidine acetate, sodium benzoate),    -   colourants (for example inorganic pigments such as, for example,        iron oxides, titanium dioxide),    -   flavourings, sweeteners, flavour- and/or odour-masking agents.

The present invention furthermore relates to a pharmaceuticalcomposition which comprise at least one compound according to theinvention, conventionally together with one or more pharmaceuticallysuitable excipient(s), and to their use according to the presentinvention.

Method of Treating Hyper-Proliferative Disorders

The present invention relates to a method for using the compounds of thepresent invention and compositions thereof, to treat mammalianhyper-proliferative disorders. Compounds can be utilized to inhibit,block, reduce, decrease, etc., cell proliferation and/or cell division,and/or produce apoptosis. This method comprises administering to amammal in need thereof, including a human, an amount of a compound ofthis invention, or a pharmaceutically acceptable salt, isomer,polymorph, metabolite, hydrate, solvate or ester thereof; etc. which iseffective to treat the disorder. Hyper-proliferative disorders includebut are not limited, e.g., psoriasis, keloids, and other hyperplasiasaffecting the skin, benign prostate hyperplasia (BPH), solid tumors,such as cancers of the breast, respiratory tract, brain, reproductiveorgans, digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid and their distant metastases. Those disorders alsoinclude lymphomas, sarcomas, and leukemias.

Examples of breast cancer include, but are not limited to invasiveductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ,and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are notlimited to small-cell and non-small-cell lung carcinoma, as well asbronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to brain stem andhypophtalmic glioma, cerebellar and cerebral astrocytoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumor.

Tumors of the male reproductive organs include, but are not limited toprostate and testicular cancer. Tumors of the female reproductive organsinclude, but are not limited to endometrial, cervical, ovarian, vaginal,and vulvar cancer, as well as sarcoma of the uterus.

Tumors of the digestive tract include, but are not limited to anal,colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal,small-intestine, and salivary gland cancers.

Tumors of the urinary tract include, but are not limited to bladder,penile, kidney, renal pelvis, ureter, urethral and human papillary renalcancers.

Eye cancers include, but are not limited to intraocular melanoma andretinoblastoma.

Examples of liver cancers include, but are not limited to hepatocellularcarcinoma (liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to laryngeal,hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oralcavity cancer and squamous cell. Lymphomas include, but are not limitedto AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-celllymphoma, Burkitt lymphoma, Hodgkin's disease, and lymphoma of thecentral nervous system.

Sarcomas include, but are not limited to sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma.

Leukemias include, but are not limited to acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

These disorders have been well characterized in humans, but also existwith a similar etiology in other mammals, and can be treated byadministering pharmaceutical compositions of the present invention.

The term “treating” or “treatment” as stated throughout this document isused conventionally, e.g., the management or care of a subject for thepurpose of combating, alleviating, reducing, relieving, improving thecondition of, etc., of a disease or disorder, such as a carcinoma.

Methods of Treating Kinase Disorders

The present invention also provides methods for the treatment ofdisorders associated with aberrant kinase activity (such as tyrosinekinase activity), including, phosphotidylinositol-3-kinase.

Effective amounts of compounds of the present invention can be used totreat disorders, including angiogenic disorders, such as cancer;inflammatory disorders (including but not limited to Chronic obstructivepulmonary disorder (COPD)), autoimmune disorders, cardiovasculardisorders (including but not limited to thrombosis, pulmonaryhypertension, cardiac hypertophy, atherosclerosis or heart failure),neurodegenerative disorders, metabolic disorders, nociceptive disorders,ophthalmic disorders, pulmonary disorders, or renal disorders.Nonetheless, such cancers and other diseases can be treated withcompounds of the present invention, regardless of the mechanism ofaction and/or the relationship between the kinase and the disorder.

The phrase “aberrant kinase activity” or “aberrant tyrosine kinaseactivity,” includes any abnormal expression or activity of the geneencoding the kinase or of the polypeptide it encodes. Examples of suchaberrant activity, include, but are not limited to, overexpression ofthe gene or polypeptide; gene amplification; mutations which produceconstitutively-active or hyperactive kinase activity; gene mutations,deletions, substitutions, additions, etc.

The present invention also provides for methods of inhibiting a kinaseactivity, especially of phosphotidylinositol-3-kinase, comprisingadministering an effective amount of a compound of the presentinvention, including salts, polymorphs, metabolites, hyrates, solvates,prodrugs (e.g.: esters) thereof, and diastereoisomeric forms thereof.Kinase activity can be inhibited in cells (e.g., in vitro), or in thecells of a mammalian subject, especially a human patient in need oftreatment.

Methods of Treating Angiogenic Disorders

The present invention also provides methods of treating disorders anddiseases associated with excessive and/or abnormal angiogenesis.

Inappropriate and ectopic expression of angiogenesis can be deleteriousto an organism. A number of pathological conditions are associated withthe growth of extraneous blood vessels. These include, e.g., diabeticretinopathy, ischemic retinal-vein occlusion, and retinopathy ofprematurity (Aiello et al. New Engl. J. Med. 1994, 331, 1480; Peer etal. Lab. Invest. 1995, 72, 638), age-related macular degeneration (AMD;see, Lopez et al. Invest. Opththalmol. Vis. Sci. 1996, 37, 855),neovascular glaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, rheumatoid arthritis (RA), restenosis, in-stentrestenosis, vascular graft restenosis, etc. In addition, the increasedblood supply associated with cancerous and neoplastic tissue, encouragesgrowth, leading to rapid tumor enlargement and metastasis. Moreover, thegrowth of new blood and lymph vessels in a tumor provides an escaperoute for renegade cells, encouraging metastasis and the consequencespread of the cancer. Thus, compounds of the present invention can beutilized to treat and/or prevent any of the aforementioned angiogenesisdisorders, e.g., by inhibiting and/or reducing blood vessel formation;by inhibiting, blocking, reducing, decreasing, etc. endothelial cellproliferation or other types involved in angiogenesis, as well ascausing cell death or apoptosis of such cell types.

Dose and Administration

Based upon standard laboratory techniques known to evaluate compoundsuseful for the treatment of hyper-proliferative disorders and angiogenicdisorders, by standard toxicity tests and by standard pharmacologicalassays for the determination of treatment of the conditions identifiedabove in mammals, and by comparison of these results with the results ofknown medicaments that are used to treat these conditions, the effectivedosage of the compounds of this invention can readily be determined fortreatment of each desired indication. The amount of the activeingredient to be administered in the treatment of one of theseconditions can vary widely according to such considerations as theparticular compound and dosage unit employed, the mode ofadministration, the period of treatment, the age and sex of the patienttreated, and the nature and extent of the condition treated.

The total amount of the active ingredient to be administered willgenerally range from about 0.001 mg/kg to about 200 mg/kg body weightper day, and preferably from about 0.01 mg/kg to about 20 mg/kg bodyweight per day. Clinically useful dosing schedules will range from oneto three times a day dosing to once every four weeks dosing. Inaddition, “drug holidays” in which a patient is not dosed with a drugfor a certain period of time, may be beneficial to the overall balancebetween pharmacological effect and tolerability. A unit dosage maycontain from about 0.5 mg to about 1500 mg of active ingredient, and canbe administered one or more times per day or less than once a day. Theaverage daily dosage for administration by injection, includingintravenous, intramuscular, subcutaneous and parenteral injections, anduse of infusion techniques will preferably be from 0.01 to 200 mg/kg oftotal body weight. The average daily rectal dosage regimen willpreferably be from 0.01 to 200 mg/kg of total body weight. The averagedaily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kgof total body weight. The average daily topical dosage regimen willpreferably be from 0.1 to 200 mg administered between one to four timesdaily. The transdermal concentration will preferably be that required tomaintain a daily dose of from 0.01 to 200 mg/kg. The average dailyinhalation dosage regimen will preferably be from 0.01 to 100 mg/kg oftotal body weight.

Of course the specific initial and continuing dosage regimen for eachpatient will vary according to the nature and severity of the conditionas determined by the attending diagnostician, the activity of thespecific compound employed, the age and general condition of thepatient, time of administration, route of administration, rate ofexcretion of the drug, drug combinations, and the like. The desired modeof treatment and number of doses of a compound of the present inventionor a pharmaceutically acceptable salt or ester or composition thereofcan be ascertained by those skilled in the art using conventionaltreatment tests.

Combination Therapies

The compounds of this invention can be administered as the solepharmaceutical agent or in combination with one or more otherpharmaceutical agents where the combination causes no unacceptableadverse effects. For example, the compounds of this invention can becombined with known anti-hyper-proliferative, antiinflammatory,analgesic, immunoregulatory, diuretic, antiarrhytmic,anti-hypercholsterolemia, anti-dyslipidemia, anti-diabetic or antiviralagents, and the like, as well as with admixtures and combinationsthereof.

The additional pharmaceutical agent can be 131I-chTNT, abarelix,abiraterone, aclarubicin, ado-trastuzumab emtansine, afatinib,aflibercept, aldesleukin, alemtuzumab, Alendronic acid, alitretinoin,altretamine, amifostine, aminoglutethimide, Hexyl aminolevulinate,amrubicin, amsacrine, anastrozole, ancestim, anethole dithiolethione,angiotensin II, antithrombin III, aprepitant, arcitumomab, arglabin,arsenic trioxide, asparaginase, axitinib, azacitidine, basiliximab,belotecan, bendamustine, belinostat, bevacizumab, bexarotene,bicalutamide, bisantrene, bleomycin, bortezomib, buserelin, bosutinib,brentuximab vedotin, busulfan, cabazitaxel, cabozantinib, calciumfolinate, calcium levofolinate, capecitabine, capromab, carboplatin,carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, celmoleukin,ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine,cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid,clofarabine, copanlisib, crisantaspase, cyclophosphamide, cyproterone,cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib,dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox,denosumab, depreotide, deslorelin, dexrazoxane, dibrospidium chloride,dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine,doxorubicin, doxorubicin+estrone, dronabinol, eculizumab, edrecolomab,elliptinium acetate, eltrombopag, endostatin, enocitabine, enzalutamide,epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta,eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine,etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim,fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide,folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant,gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide,gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine,gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron,granulocyte colony stimulating factor, histamine dihydrochloride,histrelin, hydroxycarbamide, 1-125 seeds, lansoprazole, ibandronic acid,ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib,imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate,interferon alfa, interferon beta, interferon gamma, iobitridol,iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole,ixabepilone, lanreotide, lapatinib, lasocholine, lenalidomide,lenograstim, lentinan, letrozole, leuprorelin, levamisole,levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine,lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol,melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate,methoxsalen, methylaminolevulinate, methylprednisolone,methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin,mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane,mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphinehydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin,naloxone+pentazocine, naltrexone, nartograstim, nedaplatin, nelarabine,neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide,nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab,octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole,ondansetron, oprelvekin, orgotein, orilotimod, oxaliplatin, oxycodone,oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin,palladium-103 seed, palonosetron, pamidronic acid, panitumumab,pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxyPEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b,pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane,perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin,pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate,polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide,ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone,procarbazine, procodazole, propranolol, quinagolide, rabeprazole,racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed,ramosetron, ramucirumab, ranimustine, rasburicase, razoxane,refametinib, regorafenib, risedronic acid, rhenium-186 etidronate,rituximab, romidepsin, romiplostim, romurtide, roniciclib, samarium(153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T,sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol,streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen,tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomabmerpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur,tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus,teniposide, testosterone, tetrofosmin, thalidomide, thiotepa,thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan,toremifene, tositumomab, trabectedin, tramadol, trastuzumab, trastuzumabemtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane,triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan,ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib,vinblastine, vincristine, vindesine, vinflunine, vinorelbine,vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres,zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.

Generally, the use of cytotoxic and/or cytostatic agents in combinationwith a compound or composition of the present invention will serve to:

(1) yield better efficacy in reducing the growth of a tumor or eveneliminate the tumor as compared to administration of either agent alone,(2) provide for the administration of lesser amounts of the administeredchemotherapeutic agents,(3) provide for a chemotherapeutic treatment that is well tolerated inthe patient with fewer deleterious pharmacological complications thanobserved with single agent chemotherapies and certain other combinedtherapies,(4) provide for treating a broader spectrum of different cancer types inmammals, especially humans,(5) provide for a higher response rate among treated patients,(6) provide for a longer survival time among treated patients comparedto standard chemotherapy treatments,(7) provide a longer time for tumor progression, and/or(8) yield efficacy and tolerability results at least as good as those ofthe agents used alone, compared to known instances where other canceragent combinations produce antagonistic effects.

EXPERIMENTAL Abbreviations and Acronyms

A comprehensive list of the abbreviations used by organic chemists ofordinary skill in the art appears in The ACS Style Guide (third edition)or the Guidelines for Authors for the Journal of Organic Chemistry. Theabbreviations contained in said lists, and all abbreviations utilized byorganic chemists of ordinary skill in the art are hereby incorporated byreference. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 67th Ed., 1986-87.

More specifically, when the following abbreviations are used throughoutthis disclosure, they have the following meanings:

-   -   acac acetylacetonate    -   Ac₂O acetic anhydride    -   AcO (or OAc) acetate    -   anhyd anhydrous    -   aq aqueous    -   Ar aryl    -   atm atmosphere    -   9-BBN 9-borabicyclo[3.3.1]nonyl    -   BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl    -   Bn benzyl    -   bp boiling point    -   br s broad singlet    -   Bz benzoyl    -   BOC tert-butoxycarbonyl    -   n-BuOH n-butanol    -   t-BuOH tert-butanol    -   t-BuOK potassium tert-butoxide    -   C Celsius    -   calcd calculated    -   CAN ceric ammonium nitrate    -   Cbz carbobenzyloxy    -   CDI carbonyl diimidazole    -   CD₃OD methanol-d₄    -   Celite® diatomaceous earth filter agent, Celite® Corp.    -   CI-MS chemical ionization mass spectroscopy    -   ¹³C NMR carbon-13 nuclear magnetic resonance    -   m-CPBA meta-chloroperoxybenzoic acid    -   d doublet    -   dd doublet of doublets    -   DABCO 1,4-diazabicyclo[2.2.2]octane    -   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCC N,N′-dicyclohexylcarbodiimide    -   DCM dichloromethane    -   DEAD diethyl azodicarboxylate    -   dec decomposition    -   DIA diisopropylamine    -   DIBAL diisobutylaluminum hydride    -   DMAP 4-(N,N-dimethylamino)pyridine    -   DME 1,2-dimethoxyethane    -   DMF N,N-dimethylformamide    -   DMSO dimethylsulfoxide    -   E entgegen (configuration)    -   EDCI or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide    -   EDCI.HCl hydrochloride    -   ee enantiomeric excess    -   EI electron impact    -   ELSD evaporative light scattering detector    -   equiv equivalent    -   ES-MS electrospray mass spectroscopy    -   EtOAc ethyl acetate    -   EtOH ethanol (100%)    -   EtSH ethanethiol    -   Et₂O diethyl ether    -   Et₃N triethylamine    -   Fmoc 9-fluorenylmethoxycarbonyl    -   GC gas chromatography    -   GC-MS gas chromatography-mass spectroscopy    -   h hour, hours    -   hex hexanes, or hexane    -   ¹H NMR proton nuclear magnetic resonance    -   HMPA hexamethylphosphoramide    -   HMPT hexamethylphosphoric triamide    -   HOBT hydroxybenzotriazole    -   HPLC high performance liquid chromatography    -   insol insoluble    -   IPA isopropylamine    -   iPrOH isopropylalcohol    -   IR infrared    -   J coupling constant (NMR spectroscopy)    -   L liter    -   LAH lithium aluminum hydride    -   LC liquid chromatography    -   LC-MS liquid chromatography-mass spectrometry    -   LDA lithium diisopropylamide    -   M mol L⁻¹ (molar)    -   m multiplet    -   m meta    -   MeCN acetonitrile    -   MeOH methanol    -   MHz megahertz    -   min minute, minutes    -   μL microliter    -   mL milliliter    -   μM micromolar    -   mol mole    -   mp melting point    -   MS mass spectrum, mass spectrometry    -   Ms methanesulfonyl    -   m/z mass-to-charge ratio    -   N equiv L⁻¹ (normal)    -   NBS N-bromosuccinimide    -   nM nanomolar    -   NMM 4-methylmorpholine    -   NMR Nuclear Magnetic Resonance    -   o ortho    -   obsd observed    -   p para    -   p page    -   pp pages    -   PdCl₂dppf        [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)    -   Pd(OAc)₂ palladium acetate    -   pH negative logarithm of hydrogen ion concentration    -   Ph phenyl    -   pK negative logarithm of equilibrium constant    -   pK_(a) negative logarithm of equilibrium constant for        association    -   PPA poly(phosphoric acid)    -   PS-DIEA Polystyrene-bound diisopropylethylamine    -   PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium        hexafluorophosphate    -   q quartet    -   rac racemic    -   R rectus (configurational)    -   R_(f) retardation factor (TLC)    -   RT retention time (HPLC)    -   rt room temperature    -   s singlet    -   S sinister (configurational)    -   t triplet    -   TBDMS, TBP tert-butyldimethylsilyl    -   TBDPS, TPS tert-butyldiphenylsilyl    -   TEA triethylamine    -   THF tetrahydrofuran    -   Tf trifluoromethanesulfonyl (triflyl)    -   TFA trifluoroacetic acid    -   TFFH Fluoro-N,N,N′,N′-tetramethylformamidinium        hexafluorophosphate    -   TLC thin layer chromatography    -   TMAD N,N,N′,N′-tetramethylethylenediamine    -   TMSCl trimethylsilyl chloride    -   Ts p-toluenesulfonyl    -   v/v volume to volume ratio    -   w/v weight to volume ratio    -   w/w weight to weight ratio    -   Z zusammen (configuration)

The percentage yields reported in the following examples are based onthe starting component that was used in the lowest molar amount. Air andmoisture sensitive liquids and solutions were transferred via syringe orcannula, and introduced into reaction vessels through rubber septa.Commercial grade reagents and solvents were used without furtherpurification. The term “concentrated under reduced pressure” refers touse of a Buchi rotary evaporator at approximately 15 mm of Hg. Alltemperatures are reported uncorrected in degrees Celsius (° C.). Thinlayer chromatography (TLC) was performed on pre-coated glass-backedsilica gel 60 A F-254 250 μm plates.

The structures of compounds of this invention were confirmed using oneor more of the following procedures.

NMR

NMR spectra were acquired for each compound and were consistent with thestructures shown.

Routine one-dimensional NMR spectroscopy was performed on either 400 or500 MHz Bruker® Avance spectrometers. The samples were dissolved indeuterated solvents. Chemical shifts were recorded on the ppm scale andwere usually referenced to TMS (tetramethylsilane).

General Preparative Methods

The particular process to be utilized in the preparation of thecompounds used in this embodiment of the invention depends upon thespecific compound desired. Such factors as the selection of the specificsubstituents play a role in the path to be followed in the preparationof the specific compounds of this invention. Those factors are readilyrecognized by one of ordinary skill in the art.

The compounds of the invention may be prepared by use of known chemicalreactions and procedures. Nevertheless, the following generalpreparative methods are presented to aid the reader in synthesizing thecompounds of the present invention, with more detailed particularexamples being presented below in the experimental section describingthe working examples.

The compounds of the invention can be made according to conventionalchemical methods, and/or as disclosed below, from starting materialswhich are either commercially available or producible according toroutine, conventional chemical methods. General methods for thepreparation of the compounds are given below, and the preparation ofrepresentative compounds is specifically illustrated in examples.

Synthetic transformations that may be employed in the synthesis ofcompounds of this invention and in the synthesis of intermediatesinvolved in the synthesis of compounds of this invention are known by oraccessible to one skilled in the art. Collections of synthetictransformations may be found in compilations, such as:

-   J. March. Advanced Organic Chemistry, 4th ed.; John Wiley: New York    (1992)-   R. C. Larock. Comprehensive Organic Transformations, 2nd ed.;    Wiley-VCH: New York (1999)-   F. A. Carey; R. J. Sundberg. Advanced Organic Chemistry, 2nd ed.;    Plenum Press: New York (1984)-   T. W. Greene; P. G. M. Wuts. Protective Groups in Organic Synthesis,    3rd ed.; John Wiley: New York (1999)-   L. S. Hegedus. Transition Metals in the Synthesis of Complex Organic    Molecules, 2nd ed.; University Science Books: Mill Valley, Calif.    (1994)-   L. A. Paquette, Ed. The Encyclopedia of Reagents for Organic    Synthesis; John Wiley: New York (1994)-   A. R. Katritzky; O. Meth-Cohn; C. W. Rees, Eds. Comprehensive    Organic Functional Group Transformations; Pergamon Press: Oxford, UK    (1995)-   G. Wilkinson; F. G A. Stone; E. W. Abel, Eds. Comprehensive    Organometallic Chemistry; Pergamon Press: Oxford, UK (1982)-   B. M. Trost; I. Fleming. Comprehensive Organic Synthesis; Pergamon    Press: Oxford, UK (1991)-   A. R. Katritzky; C. W. Rees Eds. Comprehensive Heterocylic    Chemistry; Pergamon Press: Oxford, UK (1984)-   A. R. Katritzky; C. W. Rees; E. F. V. Scriven, Eds. Comprehensive    Heterocylic Chemistry II; Pergamon Press: Oxford, UK (1996)-   C. Hansch; P. G. Sammes; J. B. Taylor, Eds. Comprehensive Medicinal    Chemistry: Pergamon Press: Oxford, UK (1990).

In addition, recurring reviews of synthetic methodology and relatedtopics include Organic Reactions; John Wiley: New York; OrganicSyntheses; John Wiley: New York; Reagents for Organic Synthesis: JohnWiley: New York; The Total Synthesis of Natural Products; John Wiley:New York; The Organic Chemistry of Drug Synthesis; John Wiley: New York;Annual Reports in Organic Synthesis; Academic Press: San Diego Calif.;and Methoden der Organischen Chemie (Houben-Weyl); Thieme: Stuttgart,Germany.

Furthermore, databases of synthetic transformations include ChemicalAbstracts, which may be searched using either CAS OnLine or SciFinder,Handbuch der Organischen Chemie (Beilstein), which may be searched usingSpotFire, and REACCS.

Synthesis of2-amino-N-{7-methoxy-8-[3-(3-oxomorpholin-4-yl)propoxy]-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}pyrimidine-5-carboxamide

Step 1: Preparation of8-(benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-amine

8-(benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-amine maybe prepared for example as per the method described in PCT patentapplication WO WO2008/070150, Example 1, Step 7.

Step 2: Preparation of2-amino-N-[8-(benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamide

36.3 g of (1H-benzotriazol-1-yloxy)(tripyrrolidin-1-yl)phosphoniumhexafluorophosphate (PyBOP) and then 32.4 ml of diisopropyl ethyl aminewere added to a mixture of 15 g of8-(benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-amine and7.1 g of 2-aminopyrimidine-5-carboxylic acid in 150 ml of DMF. Thereaction mixture was stirred at room temperature for 40 hours, and thenwas filtered. The collected solids were washed with ethyl acetate, anddried in vacuum to yield 17.4 g of the desired product which wasdirectly used in the next step.

Step 3: Preparation of2-amino-N-(8-hydroxy-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl)pyrimidine-5-carboxamidebis(trifluoroacetate)

84.4 ml of trifluoroacetic acid (TFA) were cooled to 0° C. 17.4 g of2-amino-N-[8-(benzyloxy)-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl]pyrimidine-5-carboxamidewere added in portions, the resulting mixture was heated up to 40° C.,and stirred at this temperature for 16 hours. The temperature was raisedto 70° C., and the mixture was stirred at this temperature foradditional 6 hours. The reaction mixture was concentrated in vacuum, andco-distilled with 30 ml dichloromethane, 30 ml of n-hexane, 60 ml of an1:1 mixture of dichloromethane:methanol (vol/vol), and finally 30 ml ofdichloromethane. 30 ml of an 1:1 mixture of n-hexane:methanol were addedto the residue, and the resulting suspension was stirred for 30 minutesat room temperature. The mixture was filtered. The collected solids werewashed twice with an 1:1 mixture of n-hexane:methanol, and the dried invacuum to yield 16.9 g. The exact trifluoroactic acid content of theproduct was not determined. The product was handled as abis(trifluoracetate).

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=13.48 (s, 1H), 12.02 (br s, 2H), 8.98(s, 2H), 7.85 (d, 1H), 7.44-7.65 (m, 2H), 7.13 (d, 1H), 4.36-4.65 (m,2H), 4.08-4.31 (m, 2H), 3.96 (s, 3H).

Step 4: Preparation of 4-(3-chloropropyl)morpholin-3-one

4.7 g of sodium hydride (60w %) were added to a mixture of 10 g ofmorpholin-3-one, 200 ml of 1,2-dimethoxymethane and 20 ml of DMF. Themixture was stirred at room temperature for 30 minutes. 18.7 g of1-bromo-3-chloropropane were added slowly using a ice-water-coolingbath. The reaction mixture was stirred at room temperature for 16 hours,and then concentrated in vacuum. The residue was dissolved in 100 ml ofdichloromethane and 100 ml of water. The layers were separated. Theorganic layer was washed three times with water, dried over sodiumsulfate, and then concentrated in vacuum. The residue was co-distilledwith 50 ml of n-heptane three times yielding 11.8 g of the desiredproduct.

¹H-NMR (400 MHz, DMSO-d₆): ϵ [ppm]=4.01 (s, 2H), 3.75-3.87 (m, 2H), 3.64(t, 2H), 3.42 (t, 2H), 3.29-3.37 (m, 2H), 1.96 (quin, 2H).

Step 5: Preparation of2-amino-N-{7-methoxy-8-[3-(3-oxomorpholin-4-yl)propoxy]-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}pyrimidine-5-carboxamide

A mixture of 16.9 g of2-amino-N-(8-hydroxy-7-methoxy-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl)pyrimidine-5-carboxamidetrifluoroacetate, 47.3 g of cesium carbonate, 10.3 g of4-(3-chloropropyl)morpholin-3-one, and 450 ml of DMF was heated up to80¹, and stirred at this temperature for 23 hours. Temperature wasraised to 100° C., and the mixture stirred at this temperature foradditional 3 hours. The mixture was cooled to room temperature, and 200ml of water were added. The mixture was stirred for one hour, and thefiltered. The collected solids were washed twice with water, and thentwice with ethanol, and dried in vacuum to yield 7.3 g of the desiredcrude product which was used without purification in the next step.

Step 6: Preparation of2-amino-N-{7-methoxy-8-[3-(3-oxomorpholin-4-yl)propoxy]-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}pyrimidine-5-carboxamidehydrochloride

Aqueous hydrochloric acid (37 w %) was added to a mixture of 7.3 g ofcrude2-amino-N-{7-methoxy-8-[3-(3-oxomorpholin-4-yl)propoxy]-2,3-dihydroimidazo[1,2-c]quinazolin-5-yl}pyrimidine-5-carboxamideas synthesized above, and 20 ml of water until pH 2 was reached. Themixture was stirred for two hours at room temperature, and then wasfiltered. n-Butanol was added to the filtrate, and the water was removedby azeotropic distillation. The resulting suspension was stirred at roomtemperature overnight, and then was filtered. The collected solids werewashed with n-heptane, and dried in vacuum to yield 4.7 g.

1H-NMR (500 MHz, DMSO-d6): δ [ppm]=13.42 (s, 1H), 12.85 (br s, 1H), 9.00(s, 2H), 8.34 (d, 1H), 7.55-7.76 (m, 2H), 7.41 (d, 1H), 4.33-4.56 (m,2H), 4.28 (t, 2H), 4.08-4.24 (m, 2H), 4.03 (s, 3H), 4.01 (s, 2H), 3.84(t, 2H), 3.54 (t, 2H), 3.40 (t, 2H), 2.07 (quin, 2H).

Biological Evaluation

The utility of the compounds of the present invention can beillustrated, for example, by their activity in vitro in the in vitrotumor cell proliferation assay described below. The link betweenactivity in tumor cell proliferation assays in vitro and anti-tumoractivity in the clinical setting has been very well established in theart. For example, the therapeutic utility of taxol (Silvestrini et al.Stem Cells 1993, 11(6), 528-35), taxotere (Bissery et al. Anti CancerDrugs 1995, 6(3), 339), and topoisomerase inhibitors (Edelman et al.Cancer Chemother. Pharmacol. 1996, 37(5), 385-93) were demonstrated withthe use of in vitro tumor proliferation assays.

Demonstration of the activity of the compounds of the present inventionmay be accomplished through in vitro, ex vivo, and in vivo assays thatare well known in the art. For example, to demonstrate the activity ofthe compounds of the present invention, the following assays may beused.

Biological Assays

The effects of the compounds of the present invention were examined bythe following assays.

[Determination of IC50 Values of Compounds in Kinase Assay of PI3Kα]Chemicals and Assay Materials

Phosphatidylinositol (Ptdlns) and phosphatidylserine (PtdSer) werepurchased from DOOSAN SERDARY RESEARCH LABORATORIES (Toronto, Canada).Recombinant truncated forms (ΔN 1-108) of the human p110α and p110βsubunits of PI3K with N-terminal His₆-Tags were expressed in S.frugiperda 9 insect cells. Recombinant human PI3Kγ (full length humanPI3K p1107 fused with a His₆-tag at the C-terminus expressed in S.frugiperda 9 insect cells) was obtained from ALEXIS BIOCHEMICALS(#201-055-C010; San Diego, Calif.). [γ³³P]ATP and unlabeled ATP werepurchased from AMERSHAM PHARMACIA BIOTECH (Buckinghamshire, UK) andROCHE DIAGNOSTICS (Mannheim, Germany), respectively. Scintillationcocktails Ultima Gold™ scintillation cocktail were purchased from PERKINELMER. Maxisorp™ plates were purchased from NALGE NUNC INTERNATIONALK.K. (Tokyo, Japan). All other chemicals not further specified were fromWAKO PURE CHEMICALS (Osaka, Japan).

Solid-Phase Lipid Kinase Assay

To assess inhibition of PI3Kα by compounds, the Maxisorp™ plates werecoated with 50 μL/well of a solution containing 50 μg/ml Ptdlns and 50μg/ml PtdSer dissolved in chloroform:ethanol (3:7). The plates weresubsequently air-dried by incubation for at least 2 hours in a fumehood. The reaction was set up by mixing 25 μL/well of assay buffer 2×(100 mM MOPSO/NaOH, 0.2 M NaCl, pH 7.0, 8 mM MgCl₂, 2 mg/mL BSA (fattyacid-free)), and 7.5 ng/well PI3Kα in the lipid pre-coated plate. 10×test compounds were added in 2% DMSO. The reaction was started by adding5 μL/well of a 40 μM ATP mix (final 10 μM ATP; 20 μCi/ml[γ³³P]ATP).After incubation at RT for 2 hours, the reaction was terminated byadding 5 μl/well stop solution (25 mM EDTA, pH 8.0). The plate was thenwashed twice with Tris-buffered saline (TBS, pH 7.4). Ultima Gold™(PERKINELMER) scintillation mix was added at 25 μL/well.

The radioactivity incorporated into the immobilized PI substrate wasdetermined with a BetaPlate Liquid Scintillation Counter (PerkinElmer).

The inhibition percent at each concentration of compound was calculated,and IC₅₀ values were determined from the inhibition of curve.

The following compounds displayed the following IC₅₀ values in theSolid-Phase Lipid p110α assay:

assay

p110α IC₅₀ (nM) 0, 7

[Isozyme Selectivity Test in PI3K] Chemicals and Assay Materials

A recombinant truncated form (ΔN 1-108) of the human p110β or p100αsubunit of PI3K with an N-terminal His₆-Tag was expressed in S.frugiperda 9 insect cells. Recombinant human PI3Kγ (full length humanPI3K p110γ fused with a His₆-tag at the C-terminus expressed in S.frugiperda 9 insect cells) was obtained fromALEXISBIOCHEMICALS(#201-055-C010; San Diego, Calif.).

Determination of IC₅₀ Values of Compounds in Kinase Assays of PI3Kβ andPI3Kγ

Kinase assays using recombinant truncated p110β or the full length p110γwere performed in a similar manner as described in the part of[Determination of IC₅₀ values of compounds in kinase assay of PI3Kα]except that these isoforms were assayed using 7.5 ng and 25.0 ng ofprotein/well, respectively.

The following compounds displayed the following IC₅₀ values in theSolid-Phase Lipid Kinase p110β assay:

assay

p110β IC₅₀ (nM) 13

[Determination of IC₅₀ Values of Compounds in Cell Based Assays of PI3KαActivity] Chemicals and Assay Materials

96-well collagen treated clear bottom/black sided Costar plates werepurchased from CORNINGLIFESCIENCES(Corning, N.Y.; at.#3904). Gibco RPMImedium (Cat.#11875), Biosource anti-phospho-AKT (Ser 473) antibody(Cat.#44-621G) and recombinant IGF-1 (Cat.# PHG0074) were purchased fromINVITROGEN(Carlsbad, Calif.). The secondary donkey anti-rabbit IgG horseradish peroxidase conjugate (Cat. # NA934V) and ECL chemiluminesencereagent (Cat.# RPN2209) were purchased from AMERSHAM (Buckinghamshire,UK). Cell culture tested bovine serum albumin solution (35% in DPBS;Cat.# A7979) and all other chemicals were purchased from SIGMA (St.Louis, Mo.). The Wallac Victor2 1420 Multilabel HTS Counter waspurchased from PERKINELMER(Wellesley, Mass.)

IGF-1 Induced AKT Phosphorylation Assay

To test inhibition of IGF-1 induced AKT phosphorylation by compounds,A549 cells (5×10⁴ cells/well) were seeded in 100 μL of 0.1% bovine serumalbumin (BSA) in RPMI medium in 96-well collagen treated clearbottom/black sided plates and incubated overnight at 37° C. in a 5% CO₂incubator. 10× compound solution (in 0.1% BSA in RPMI) was added to theplates and incubation at 37° C. was continued for 1 hour. All wells(except no IGF-1 controls) were then treated with 25 ng/ml IGF-1 for 10minutes at 37° C. in a 5% CO₂ incubator. Following removal of thesupernatants and washing with the wells with TBS (50 mM Tris pH 8.0containing 138 mM NaCL and 27 mM KCl), 200 μL of 3.7% formaldehyde inTBS was added to each well, and the plate was incubated at 4° C. for 10minutes. Supernatants were once again removed and replaced with 50 μLMethanol (−20° C.) and the plate incubated at 4° C. f or 5 minutes. 200μL of 0.1% BSA in TBS was then added to each well and the plateincubated at room temperature for ½ hour. Supernatants were removed and50 μL of a solution comprising the primary anti-phospho-AKT (Ser 473)antibody diluted 1:250 in TBS containing 0.1% BSA was added to each well(except control/background wells). The plate was then incubated for 1½hour at room temperature. Supernatants were removed, each well waswashed 3 times with 200 μL TBS, and 100 μL of a solution containing thesecondary donkey anti-rabbit IgG antibody HRP-conjugate diluted 1:100 inTBS-T (TBS containing 0.1% triton). Plates were then incubated for 1hour at room temperature. After removing the secondary antibody, eachwell was washed 6 times with cold TBS-T, 100 μL of ECL was added to eachwell, and the plate was placed on an orbital shaker for 1 minute. Theplates were then read on a Wallac Victor2 1420 Multilabel HTS Counterusing the luminometry window (maximum light detection is measured at 428nM). IC₅₀ values were determined from the inhibition curve.

Mouse

To evaluate the in vivo anti-tumor effect of PI3K inhibitors, efficacystudies were conducted in the NCr athymic female mice (Taconic, N.Y.).Human carcinoma cells of various histological types were harvested frommid-log phase cultures using Trypsin-EDTA (Gibco). Cells were pelleted,rinsed twice, and resuspended in sterile HBSS (Hank's Balanced SaltSolution) to final concentration of 2.5×10⁶ cells/ml. Cells wereimplanted subcutaneously (s.c.) in a 0.2 ml volume (5×10⁶ cells) intothe right flank. When tumors reached an average size of ˜100-125 mg, themice were randomized, and treatment initiated. Each experimental groupconsisted of 10 mice and the dosing volume was 10 ml/kg body weight.Compounds were dissolved in a compatible vehicle for both intravenousand oral administration. For intravenous administration, mice are placedunder a heat lamp to warm for 5 minutes, then placed in a restrainingdevice and the tail vein injected with a sterile 27 gauge ½ inch needle.Oral dosing utilizes sterile disposable feeding needles (20 gauge/1½inches) from Popper and Sons, New Hyde Park, N.Y. Tumor growth wasmeasured with electronic calipers 2-3 times a week and tumor weight (mg)calculated according to the following formula: [length (mm)×width(mm)2]/2. Percent inhibition or tumor growth inhibition (TGI) iscalculated on days of measurement using the following formula: (100−meantumor value of treated (T)/mean tumor of control value (C)×100)=% T/C.Of note: the control used in the calculations is either the “untreatedcontrol” or “vehicle”, whichever provides the most conservativerepresentation of the data.

Rat

To evaluate the in vivo anti-tumor effect of PI3K inhibitors, efficacystudies were conducted in the HSD athymic female rats (Harlan, ID).Human carcinoma cells of various histological types were harvested frommid-log phase cultures using Trypsin-EDTA (Gibco). Cells were pelleted,rinsed twice, and resuspended in sterile HBSS (Hank's Balanced SaltSolution) to final concentration of 2.5×106 cells/ml. Cells wereimplanted subcutaneously (s.c.) in a 0.2 ml volume (5×106 cells) intothe right flank. When tumors reached an average size of ˜200-400 mg, therats were randomized, and treatment initiated. Each experimental groupconsisted of 10 nude rats. Compounds were dissolved in a compatiblevehicle for both intravenous and oral administration. For intravenousadministration of compound, rats were warmed under a heating lamp for 5minutes, then placed in a restraining device, and injected intravenouslyvia the tail vein using a dosing volume ranging from 2 mL/kg to 5 mL/kgwith a sterile 25 gauge needle. Oral dosing utilizes sterile disposablefeeding needles (18 gauge/2 inch) from Popper and Sons, New Hyde Park,N.Y. Tumor growth was measured with electronic calipers 2-3 times a weekand tumor weight (mg) calculated according to the following formula:[length (mm)×width (mm)2]/2. Percent inhibition or tumor growthinhibition (TGI) is calculated on days of measurement using thefollowing formula: (100−mean tumor value of treated (T)/mean tumor ofcontrol value (C)×100)=% T/C. Of note: the control used in thecalculations is either the “untreated control” or “vehicle”, whicheverprovides the most conservative representation of the data.

It is believed that one skilled in the art, using the preceedinginformation and information available in the art, can utilize thepresent invention to its fullest extent. Those skilled in the art willrecognize that the invention may be practiced with variations on thedisclosed structures, materials, compositions and methods withoutdeparting from the spirit or scope of the invention as it is set forthherein and such variations are regarded as within the ambit of theinvention. The compounds described in the examples are intended to berepresentative of the invention, and it will be understood that thescope of the invention is not limited by the scope of the examples. Thetopic headings set forth above are meant as guidance where certaininformation can be found in the application, but are not intended to bethe only source in the application where information on such topics canbe found. All publications and patents cited above are incorporatedherein by reference.

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1: A compound of formula (I):

wherein: R¹ is a hydrogen atom or a —C(═O)H group; and R^(1′) is a—(CH₂)₂OH group; or: R¹ and R^(1′), together with the N-atom to whichthey are attached, form a morpholinyl group which is substituted with 1or 2 substituents independently selected from —OH and ═O, or aphysiologically acceptable salt, solvate, hydrate or stereoisomerthereof. 2: The compound according to claim 1, wherein: R¹ is a hydrogenatom; and R^(1′) is a —(CH₂)₂OH group, or a physiologically acceptablesalt, solvate, hydrate or stereoisomer thereof. 3: The compoundaccording to claim 1, wherein: R¹ is a —C(═O)H group; and R^(1′) is a—(CH₂)₂OH group, or a physiologically acceptable salt, solvate, hydrateor stereoisomer thereof. 4: The compound according to claim 1, wherein:R¹ and R^(1′), together with the N-atom to which they are attached, forma morpholinyl group which is substituted with 1 or 2 substituentsindependently selected from —OH and ═O, or a physiologically acceptablesalt, solvate, hydrate or stereoisomer thereof. 5: The compoundaccording to claim 1, wherein: R¹ and R^(1′), together with the N-atomto which they are attached, form a morpholinyl group which issubstituted with a —OH and a ═O substituent, or a physiologicallyacceptable salt, solvate, hydrate or stereoisomer thereof. 6: Thecompound according to claim 1, wherein: R¹ and R^(1′), together with theN-atom to which they are attached, form a morpholinyl group which issubstituted with a —OH substituent, or a physiologically acceptablesalt, solvate, hydrate or stereoisomer thereof. 7: The compoundaccording to claim 1, wherein: R¹ and R^(1′), together with the N-atomto which they are attached, form a morpholinyl group which issubstituted with a ═O substituent, or a physiologically acceptable salt,solvate, hydrate or stereoisomer thereof. 8: The compound according toclaim 1, wherein: R¹ and R^(1′), together with the N-atom to which theyare attached, form a morpholinyl group which is substituted with a ═Osubstituent, wherein said ═O group is bound to a carbon atom adjacent tosaid N-atom, or a physiologically acceptable salt, solvate, hydrate orstereoisomer thereof. 9: The compound according to claim 1, wherein thecompound is:

or a physiologically acceptable salt thereof. 10: The compound accordingto claim 1, wherein the compound is:

or a physiologically acceptable salt thereof. 11: The compound accordingto claim 1, wherein the compound is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof. 12: The compound according to claim 1, wherein the compound is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof. 13: The compound according to claim 1, wherein the compound is:

or a physiologically acceptable salt, solvate, hydrate or stereoisomerthereof. 14: A pharmaceutical composition comprising a compound offormula (I) according to claim 1, or a physiologically acceptable salt,solvate, hydrate or stereoisomer thereof, and a pharmaceuticallyacceptable diluent or carrier. 15: The pharmaceutical compositionaccording to claim 14, wherein said compound is present in atherapeutically effective amount. 16: The pharmaceutical compositionaccording to claim 15, which further comprises at least one furtheractive compound. 17: The pharmaceutical composition of claim 16, whereinthe further active compound is an anti-hyper-proliferative,anti-inflammatory, analgesic, immunoregulatory, diuretic,anti-arrhythmic, anti-hypercholesterolemic, anti-diabetic,anti-dyslipidemia, anti-diabetic or antiviral agent. 18: Thepharmaceutical composition of claim 17, wherein the further activecompound is 131I-chTNT, abarelix, abiraterone, aclarubicin,ado-trastuzumab emtansine, afatinib, aflibercept, aldesleukin,alemtuzumab, Alendronic acid, alitretinoin, altretamine, amifostine,aminoglutethimide, Hexyl aminolevulinate, amrubicin, amsacrine,anastrozole, ancestim, anethole dithiolethione, angiotensin II,antithrombin III, aprepitant, arcitumomab, arglabin, arsenic trioxide,asparaginase, axitinib, azacitidine, basiliximab, belotecan,bendamustine, belinostat, bevacizumab, bexarotene, bicalutamide,bisantrene, bleomycin, bortezomib, buserelin, bosutinib, brentuximabvedotin, busulfan, cabazitaxel, cabozantinib, calcium folinate, calciumlevofolinate, capecitabine, capromab, carboplatin, carfilzomib,carmofur, carmustine, catumaxomab, celecoxib, celmoleukin, ceritinib,cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir,cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine,copanlisib, crisantaspase, cyclophosphamide, cyproterone, cytarabine,dacarbazine, dactinomycin, darbepoetin alfa, dabrafenib, dasatinib,daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab,depreotide, deslorelin, dexrazoxane, dibrospidium chloride,dianhydrogalactitol, diclofenac, docetaxel, dolasetron, doxifluridine,doxorubicin, doxorubicin+estrone, dronabinol, eculizumab, edrecolomab,elliptinium acetate, eltrombopag, endostatin, enocitabine, enzalutamide,epirubicin, epitiostanol, epoetin alfa, epoetin beta, epoetin zeta,eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine,etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim,fluoxymesterone, floxuridine, fludarabine, fluorouracil, flutamide,folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant,gadobutrol, gadoteridol, gadoteric acid meglumine, gadoversetamide,gadoxetic acid, gallium nitrate, ganirelix, gefitinib, gemcitabine,gemtuzumab, Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron,granulocyte colony stimulating factor, histamine dihydrochloride,histrelin, hydroxycarbamide, I-125 seeds, lansoprazole, ibandronic acid,ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib,imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate,interferon alfa, interferon beta, interferon gamma, iobitridol,iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole,ixabepilone, lanreotide, lapatinib, lasocholine, lenalidomide,lenograstim, lentinan, letrozole, leuprorelin, levamisole,levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine,lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol,melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate,methoxsalen, methylaminolevulinate, methylprednisolone,methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin,mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane,mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphinehydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin,naloxone+pentazocine, naltrexone, nartograstim, nedaplatin, nelarabine,neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide,nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab,octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole,ondansetron, oprelvekin, orgotein, orilotimod, oxaliplatin, oxycodone,oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin,palladium-103 seed, palonosetron, pamidronic acid, panitumumab,pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxyPEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b,pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane,perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin,pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate,polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide,ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone,procarbazine, procodazole, propranolol, quinagolide, rabeprazole,racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed,ramosetron, ramucirumab, ranimustine, rasburicase, razoxane,refametinib, regorafenib, risedronic acid, rhenium-186 etidronate,rituximab, romidepsin, romiplostim, romurtide, roniciclib, samarium(153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T,sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol,streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen,tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomabmerpentan, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur,tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus,teniposide, testosterone, tetrofosmin, thalidomide, thiotepa,thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan,toremifene, tositumomab, trabectedin, tramadol, trastuzumab, trastuzumabemtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane,triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan,ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib,vinblastine, vincristine, vindesine, vinflunine, vinorelbine,vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres,zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin, orcombinations thereof. 19: A packaged pharmaceutical compositioncomprising a container, the pharmaceutical composition of claim 14, andinstructions for using the pharmaceutical composition to treat a diseaseor condition in a mammal. 20: A method of inhibitingphosphotidylinositol-3-kinase in cells comprising contacting a cell withone or more compounds according to claim
 1. 21: A method of treating adisorder mediated by phosphotidylinositol-3-kinase in a mammal,comprising administering to a mammal in need thereof, a therapeuticallyeffective amount of one or more compounds according to claim
 1. 22: Themethod of claim 21, wherein said disorder mediated byphosphotidylinosito-3-kinase is an angiogenic disorder, an inflammatorydisorder, an autoimmune disorder, a cardiovascular disorder, aneurodegenerative disorder, a metabolic disorder, a nociceptivedisorder, an ophthalmic disorder, a pulmonary disorder, or a renaldisorder. 23: The method of claim 22, wherein the cardiovasculardisorder is thrombosis, pulmonary hypertension, cardiac hypertophy,atherosclerosis or heart failure. 24: The method of claim 22, whereinthe inflammatory disorder is COPD. 25: The method of claim 22, whereinthe angiogenic disorder diabetic retinopathy, ischemic retinal-veinocclusion, retinopathy of prematurity, macular degeneration, neovascularglaucoma, psoriasis, retrolental fibroplasias, angiofibroma,inflammation, rheumatoid arthritis, restenosis, in-stent restenosis, orvascular graft restenosis. 26: A method of treating a hyperproliferativedisorder in a mammal, comprising administering to a mammal in needthereof, a therapeutically effective amount of one or more compoundsaccording to claim
 1. 27: The method of claim 26, wherein thehyperproliferative disorder is cancer. 28: The method of claim 27,wherein the cancer is a cancer of the breast, respiratory tract, brain,reproductive organs, digestive tract, urinary tract, eye, liver, skin,head and neck, thyroid, parathyroid or a distant metastasis of a solidtumor. 29: The method of claim 27, wherein the cancer is a lymphoma,sarcoma, or leukemia. 30-33. (canceled) 34: The method of claim 28,wherein said cancer of digestive tract is colorectal cancer. 35: Themethod of claim 28, wherein said cancer of respiratory tract isnon-small cell lung carcinoma. 36: The method of claim 29, wherein saidlymphoma is non-Hodgkin's lymphoma.